Small animal fluorescence tomography has emerged as powerful biomedical research tool since it allows three-dimensional imaging of molecularly-targeted fluorescent probes and red-shifted fluorescent proteins in unperturbed environments in vivo. Despite significant advances in instrumentation and image reconstruction algorithms in recent years, fluorescence tomographic imaging technology remains far from optimized. Two critical limitations are the relatively poor image resolution (limited to 1 or 2 mm) due to the high degree of light scatter in biological tissues, and an inability to perform high-throughput imaging of many fluorescent targets simultaneously (i.e. multiplexing), largely due to the spectral overlap of common fluorophores, long data acquisition times and tissue autofluorescence. In this project, we will develop a highly novel fluorescence tomographic scanner that will address these critical limitations using a number of unique design elements including; i) high- speed time-gated photon counting detection of the earliest-transmitted and therefore least scattered photons through animals, thereby allowing imaging resolution approaching 250 5m without loss of sensitivity or accuracy, ii) fast acquisition of both hyperspectral and fluorescence lifetime data in about 1 minute per axial slice, allowing robust de-mixing of at least five concurrent fluorophores and rejection of tissue autofluorescence, iii) a high speed, pulsed supercontinuum light source for excitation of virtually any fluorophore in the red and near-infrared region. The system will image mice in sequential axial slices over 360 degrees in an instrument configuration analogous to X-ray Computed Tomography. We will demonstrate that the scanner is capable of high-resolution multiplexed imaging, first in complex fluorescent optical phantoms with simulated background autofluorescence, and secondly in nude mice with multiply-labeled human glioma (Gli-36 and GBM8) tumor xenografts. We anticipate that the system will have applications in many areas of biomedical research including studying disease development and response to novel therapeutics non- invasively in live animals. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a highly novel fluorescence tomographic imager for high- resolution multiplexed imaging of molecular probes and red-shifted fluorescent proteins in whole animals in vivo. The unique design of the scanner will allow rapid, concurrent acquisition of hyperspectral and temporal data sets in an optical instrument configuration analogous to X-ray Computed Tomography. Novel image reconstruction algorithms will allow robust de-mixing and visualization of at least five fluorescent constructs with a resolution close to 250 5m, offering unprecedented small animal imaging capabilities. We anticipate that the scanner will have many applications in biomedical research including studying disease development and response to novel therapeutics in vivo.